Abstract

Introduction: The CpG island methylator phenotype (CIMP) is a distinct phenotype associated with microsatellite instability (MSI) and BRAF mutation in colon cancer. Recent investigations have selected 5 promoters (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1) as surrogate markers for CIMP-high. However, no study has comprehensively evaluated an expanded set of methylation markers (including these 5 markers) using a large number of tumors, or deciphered the complex clinical and molecular associations with CIMP-high determined by the validated marker panel. Materials and Methods: DNA methylation at 16 CpG islands [the above 5 plus CDKN2A (p16), CHFR, CRABP1, HIC1, IGFBP3, MGMT, MINT1, MINT31, MLH1, p14 (CDKN2A/ARF) and WRN] was quantified in 904 colorectal cancers by real-time PCR (MethyLight). To evaluate 16 methylation markers in an unbiased fashion, we conducted an unsupervised hierarchical clustering analysis of the 16 methylation markers and status of MSI, and KRAS and BRAF oncogenes. In order to accurately quantify relatively high LINE-1 methylation levels, we utilized Pyrosequencing. Results: In unsupervised hierarchical clustering analysis, the 5 markers (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1), CDKN2A, CRABP1, MINT31, MLH1, p14 and WRN were generally clustered with each other and with MSI and BRAF mutation. KRAS mutation was not clustered with any methylation marker, suggesting its association with a random methylation pattern in CIMP-low tumors. Utilizing the validated CIMP marker panel (including the above 5 markers), multivariate logistic regression analysis demonstrated that CIMP-high was independently associated with older age, proximal location, poor differentiation, MSI-high, BRAF mutation, and inversely with LINE-1 hypomethylation and \#946;-catenin activation. Mucinous feature, signet ring cells, and p53-negativity were associated with CIMP-high in only univariate analysis. In stratified analyses, the relations of CIMP-high with poor differentiation, KRAS mutation and LINE-1 hypomethylation significantly differed according to MSI status. Conclusion: Using the 16 methylation markers and a large population-based sample, we have evaluated performance of each of the 16 methylation markers in an unbiased fashion. Our current study provides valuable data for standardization of the use of CIMP-high-specific markers. Using the validated CIMP-specific methylation marker panel, we have comprehensively analyzed the clinical, pathologic and molecular features of CIMP-high colorectal cancer by comprehensive biostatistical methods. We have provided the rationale to use the validated CIMP-high-specific methylation marker panel in clinical and research settings. Our data also suggest that KRAS mutation is related with a random CpG island methylation pattern, leading to CIMP-low tumors.